Supinoxin blocks Small Cell Lung Cancer Progression by Inhibiting Mitochondrial Respiration through the RNA Helicase DDX5

oxidative phosphorylation. Thus, Supinoxin is a new therapeutic agent for small cell lung cancer (SCLC)


Introduction
DDX5 belongs to the DEAD-box RNA helicase family, which is the largest class of RNAdependent helicases found in all forms of life 14 .DEAD-box proteins exhibit both RNAdependent ATPase activity and ATP-dependent helicase activity 15 .DEAD-box proteins are involved in various stages of a cellular RNAs' lifespan, including transcription, pre-mRNA processing, RNA degradation, and ribosome biogenesis [15][16][17][18] .Despite identification of various roles of DEAD-box proteins in vitro, specific mechanisms of action in vivo have remained elusive.Results from our lab indicate that DDX5 exhibits RNA helicase activity in vitro and shares functional similarities with its counterpart DBP2 in Saccharomyces cerevisiae 19 .Deleting DBP2 in S. cerevisiae results in significant changes to mRNA secondary structures, which in turn affect transcriptional termination 16 .In addition, previous studies have demonstrated that DDX5 plays a role in regulating alternative splicing through potential secondary structure remodeling 20,21 .It appears that DDX5 and Dbp2 are responsible for the remodeling of RNA secondary structures in nascent RNA during the transcription and pre-mRNA maturation phases, which most likely contributes to the regulation of gene expression.
The DEAD-box family members have been linked to various human diseases, including neurological disorders and cancers 1,22 .DDX5 overexpression has been associated with a range of cancers, such as breast cancer, colon cancer, lung cancer, prostate cancer, and others [1][2][3][4]18 . DDX also serves as a cofactor for oncogenic transcription factors in various cancer types 4,23 .The expression of DDX5 activates the oncogenic Wnt and mammalian target of rapamycin (mTOR) signaling pathways, which play crucial roles in cell-fate determination and cell growth [24][25][26] .The phosphorylation of DDX5 at tyrosine 593 (Y593) plays a crucial role in promoting epithelial-mesenchymal transition (EMT) and facilitating the nuclear translocation of β-catenin in colon cancer cell lines 27,28 .Phosphorylation of DDX5 at Y593 has been demonstrated to activate the transcription of Cyclin D1 and c-Myc, as well as promote cell proliferation. Thes findings provide additional evidence supporting the oncogenic role of DDX5 29 .Supinoxin, also referred to as RX-5902, is a compound that has been suggested to target pDDX5, a phosphorylated form of DDX5, with potential anticancer effects 12,13 .The compound's anticancer properties were initially discovered during a screening of novel quinoxalinyl-piperazine compounds 11 .In a study conducted in 2010, RX-5902 (referred to as compound 25) demonstrated encouraging IC50 values in inhibiting the growth of various cancer cell lines, including MDA-MB-231 cells, which are a type of breast adenocarcinoma human cell lines 11 .Studies conducted on rats have shown that Supinoxin has promising bioavailability properties, making it a potential candidate for the development of an anticancer drug 11,30 . In 205, a significant discovery was made regarding the connection between Supinoxin and DDX5.It was found that Supinoxin specifically binds to Y593 phosphorylated DDX5 13 .
Importantly, Supinoxin also shown to decrease expression levels of p-c-Jun, c-Myc, and Cyclin D1 which reportedly are regulated by a novel pDDX5/β-catenin interaction 13,31 .Thus, it was suggested that Supinoxin acts by interfering with the interaction between pDDX5 and β-catenin, resulting in decreased expression of β-catenin-dependent genes 13 .Data from molecular dynamics simulation assays suggest that Supinoxin binding to pDDX5 could potentially lead to conformational changes in pDDX5 which could preclude binding of β-catenin to pDDX5 32 .A decrease of nuclear β-catenin and the βcatenin-dependent gene MCL-1 has also been shown in upon Supinoxin treatment in triple negative breast cancer (TNBC) cell lines 12 .The MDA-MB-231 xenograft model showed a significant reduction in pDDX5, c-Myc, and β-catenin protein levels after treatment with Supinoxin 12 .Thus, the model proposed is that Supinoxin interferes with the interaction between pDDX5 and β-catenin, precluding the nuclear translocation of βcatenin and the expression of β-catenin-dependent genes.Data from our lab shows that DDX5 is upregulated in small cell lung cancer (SCLC), an extremely aggressive and lethal, recalcitrant tumor 5 .Despite being the sixth-most common cause of cancer-related deaths worldwide, the unfortunate reality is that the life expectancy for patients diagnosed with SCLC remains short and treatment options have seen little improvement over the past three decades 33 .The current treatment involves a combination of etoposide/platinum-based chemotherapy and anti-programmed deathligand 1 (anti-PD-L1) immunotherapy.Sadly, the majority of these patients relapse and only 10-20% survive beyond 2 years, with a median survival time from 7 to 12 months 12,[33][34][35][36] .Previously, our lab has shown that DDX5 knockdown reduces proliferation and soft agar colony formation of human SCLC 5 .Furthermore, our lab has shown through RNA-seq and metabolic assays that mitochondrial function is dysregulated upon DDX5 knockdown 5 .Together, these data implicate DDX5 in playing a role in SCLC growth via respiration.
Herein, we show that there is a significant overexpression of DDX5 in SCLC cell lines, primarily attributed to increased stability of DDX5 proteins in these cells.Moreover, significant DDX5 expression has been observed in patient-derived xenograft (PDX) SCLC tumors, indicating its involvement in SCLC development.In addition, we observed that Supinoxin (RX-5902) is highly effective in inhibiting the growth of small cell lung cancer (SCLC) cells both in vitro SCLC cell lines and in vivo mouse models.
Next, we aimed to investigate the molecular mechanism by which Supinoxin affects SCLC.Several insights from our lab indicate that the current model of Supinoxin activity may not be accurate.Rather, Supinoxin impedes the activity of genes linked to oxidative phosphorylation, resulting in mitochondrial dysfunction in SCLC cell lines.

DDX5 protein is more stable in SCLC cell lines.
Previous studies conducted in our lab have shown that the levels of DDX5 protein is significantly higher in H69 (chemosensitive) and H69AR (chemoresistant) cell lines in comparison to the bronchial epithelial cell line HBEC-3KT (HBEC) 5 .DDX5 has been found to play a role in the progression of different types of cancers 24,37,38 .In order to validate the results of our previous study, we conducted Western blot analyses to assess the levels of DDX5 proteins in three cell lines: H69 (chemosensitive) and H69AR (chemoresistant) SCLC cell lines, as well as the noncancerous human bronchial epithelial cell line HBEC-3KT (HBEC).We utilized a different monoclonal antibody specific to DDX5 for this study, as the level of DDX5 expression in normal lung epithelial HBEC cells is below the detection threshold in our previous study 5 .The results indicate that the DDX5 protein is significantly overexpressed in SCLC cell lines compared to the noncancerous human bronchial epithelial cell line HBEC-3KT (Figure 1A-B).The levels of RFC1 protein were comparable in both small cell lung cancer (SCLC) lines and the noncancerous bronchial epithelial cell line HBEC-3KT (Figure 1A-B).Furthermore, we tried to investigate the cause of upregulation of DDX5 in SCLC cell lines.Previous research 39 (data obtained from Gene Expression Omnibus (GEO): GDS4794/225886_at) of 23 clinical small cell lung cancer (SCLC) samples from patients undergoing pulmonary resection revealed that DDX5 gene expression is actually downregulated in comparison to a normal individual (Figure 1C).Moreover, the data set from Gene Expression Profiling Interactive Analysis (GEPIA) (ENSG00000108654) indicates that DDX5 expression is downregulated or has remained at similar levels in the majority of tumor samples and paired normal tissues (Figure 1D).In this study, we aimed to determine if the elevated levels of DDX5 transcripts in SCLC cell lines are the cause of the DDX5 protein overexpression in SCLC cell lines.It has been observed that the levels of DDX5 transcripts are significantly downregulated in both H69 (chemosensitive) and also relatively similar in H69AR (chemoresistant) SCLC cell lines compared to the noncancerous human bronchial epithelial cell line HBEC-3KT (Figure 1E).These data are consistent with the prior datasets from (GEO) (GDS4794/225886_at) and (GEPIA) (ENSG00000108654), respectively.Therefore, a clear inverse relationship can be observed between the expression of DDX5 mRNA and its protein levels.We then determined the relative stability of DDX5 protein using cycloheximide (CHX) chase assays in H69 (chemosensitive), H69AR (chemoresistant), and noncancerous human bronchial epithelial cell line HBEC-3KT cell lines (Figure 1F-H).CHX hinders protein synthesis by blocking the elongation phase of protein translation.In comparison to the human bronchial epithelial cell line HBEC-3KT, it was shown that DDX5 is significantly more stable after treatment with CHX in both the chemosensitive H69 and the chemoresistant H69AR SCLC cell lines.On the other hand, the relative stability of RFC1 remains comparable across all the cell lines (Figure 1F-J).Thus, the increased stability of DDX5 protein, rather than the increase in DDX5 transcripts is responsible for the observed overexpression in SCLC.

Supinoxin inhibits the growth of H69AR cells.
In 2015, the Kost lab discovered the initial link between Supinoxin and DDX5 13 .Through a drug affinity responsive target stability experiment (DARTS) and a filter binding assay, they found that Supinoxin specifically binds to Y593 phosphorylated DDX5, identifying DDX5 as a cellular target of Supinoxin 13 .Data from our lab shows that, just like triple negative breast cancer cells (TNBC), DDX5 is also upregulated in small cell lung cancer (SCLC), an extremely aggressive and lethal, recalcitrant tumor 5 .
Based on previous studies, our lab strived to identify whether Supinoxin could potentially be repurposed for treatment of SCLC.To answer this question, we first examined the effects of Supinoxin on H69AR cells, an Adriamycin-resistant adherent SCLC cell line.We found that Supinoxin inhibits the proliferation of H69AR cell lines, with IC50 value 65.7 ±10.3 (Mean ± SD). (Figure 2A).Next, we asked whether Supinoxin inhibits anchorage-independent growth in SCLC, a hallmark of carcinogenesis.We measured the formation of H69AR colonies in soft agar in the absence and presence of 70nM of Supinoxin.Interestingly, Supinoxin was able to inhibit colony formation of H69AR cells in soft agar (Figure 2B).Based on these results, it is evident that Supinoxin inhibits the growth of H69AR cells.

Supinoxin inhibits H69AR based tumors patient-derived xenograft (PDX) SCLC tumors in mice.
Next, we investigated the effect of Supinoxin on SCLC in vivo by asking if Supinoxin can reduce the growth of H69AR xenograft tumors in vivo.To test this, immunocompromised mice with H69AR xenograft tumors were treated with different concentrations of Supinoxin (17.5, 35, 70 mg/kg).Our results demonstrate significant tumor growth inhibition (TGI) of SCLC tumors in mice with the most striking results observed at 70mg/kg dosage.(Figure 3A).Additionally, we evaluated the expression of DDX5 in Patient Derived Xenograft (PDX) Small Cell Lung Cancer samples (SCLCs).Immunohistochemical (IHC) staining revealed a significant upregulation of DDX5 in patient-derived xenograft (PDX) SCLC tumors compared to normal lung tissue.(Figure 3B).To test Supinoxin in conditions more representative of actual cancer patients, we collaborated with Purdue University's Biological Evaluation Shared Resource (BESR) to create a patient-derived xenograft tumor model using a xenograft from Jackson Laboratory (JAX).We tested the effects of 70mg/kg Supinoxin on tumor growth in immunocompromised mice with SCLC PDX tumors versus a vehicle-only control.
Results showed 70mg/kg of Supinoxin treatment was sufficient to inhibit tumor growth and improve survival of the mice with SCLC PDX tumors (Figure 3C).We stopped the drug treatment after 8 weeks to see if Supinoxin permanently inhibited tumor growth.
Interestingly, Supinoxin treatment resulted in tumor growth inhibition (TGI), with significant difference at the concentration tested (70mg/kg) compared with vehicle (Figure 3D).

The expression of c-Myc, DDX5 and β-Catenin remained unaltered upon treatment Supinoxin.
Previous data from other labs suggested that Supinoxin acts by interfering with the interaction of pDDX5 and β-catenin, resulting in decreased expression of β-catenindependent genes like c-Myc, Cyclin D1, etc. responsible for cancer progression in MDA-MB-231, a triple negative breast cancer cell line 12 .To independently validate previous claims that treatment of Supinoxin decreases the protein levels of β-catenin-dependent genes, we performed Supinoxin treatment on H69AR cells using the same conditions (0, 20, and 70nM incubation for 24hrs) described in Kost et al. 13 .Much to our surprise, western blotting showed no significant change in the protein levels of c-Myc, a βcatenin-dependent gene, upon Supinoxin treatment.We also did not observe any significant change in DDX5 or β-Catenin protein levels upon Supinoxin treatment (Figure 4B-E).Subsequently, we analyzed the protein levels of c-Myc, DDX5 and β-Catenin upon Supinoxin treatment in MDA-MB-231 cells.This revealed that the levels of c-Myc, DDX5 and β-Catenin proteins remained unaltered upon Supinoxin treatment.
(Figure 5B-E).We then determined the relative steady-state levels of c-Myc, Cyclin-D1

The localization of β-Catenin and pDDX5 remains unaffected upon treatment with Supinoxin.
Data suggest 12,13,40 that Supinoxin treatment results in a decrease in nuclear β -Catenin levels.To test this, we performed immunofluorescence microscopy on MDA-MB-231 and H69AR cells treated for 24 hours with (70nM) and without Supinoxin and found no significant difference in nuclear β-Catenin localization in both the conditions (Figure 6A).Given that Supinoxin is thought to specifically target Y593 phosphorylated DDX5 (pDDX5) and that Supinoxin treatment has no effect on DDX5 protein levels, we investigated whether Supinoxin treatment alters the localization of pDDX5 in H69AR and MDA-MB-231 cells.pDDX5 is primarily cytoplasmic, in contrast to the total DDX5, which is predominantly nuclear.However, Supinoxin treatment of H69AR and MDA-MB-231 cells resulted in no significant changes in pDDX5 localization (Figure 6B).Next, we investigated the localization of pDDX5 at various time points upon treatment with 100 mM of Supinoxin in MDA-MB-231 cells.Again, no significant change in pDDX5 localization was observed over time after Supinoxin treatment (Figure 6C).

Genes involved in mitochondrial function are downregulated in H69AR cells upon Supinoxin treatment.
In order to identify the genes impacted by Supinoxin treatment on a global level, RNA-Seq was conducted on H69AR cells treated with or without Supinoxin.We obtained approximately 2 × 10 8 mapped reads for each of the three biological replicates.The genome mapping rates ranged from 60% to 95%.The separation of samples by their group and the localization of samples within groups were visualized using DESeq2's plot Principal Components Analysis (PCA) function.We chose to use three replicates of H69AR, both with or without Supinoxin, based on the variance.The PCA algorithm offers a comprehensive analysis of the primary directions of greatest variability in the data, making it suitable for clustering purposes.The data analysis involved the use of DESeq2 and edgeR, which successfully identified a significant number of differentially expressed genes (DEGs) upon the addition of Supinoxin with a false discovery rate (FDR) of < 0.05.A total of 455 transcripts (Figure 7A) are significantly downregulated upon both Supinoxin treatment and DDX5 knockdown 5 obtained from our previous work.The gene expression data was analyzed and mapped to the KEGG pathway to investigate the activation and inhibition of different DEGs (Figure 7B-C) across various classes of pathways.Pathway analysis and functional annotation for up-and downregulated genes were performed using the R software package enrichR and GSEA.A total of 1486 genes were found to be up-regulated, while 1456 genes were downregulated.These genes were mapped to a total of 200 KEGG pathways.Figure 7D displays the top 10 enriched pathways.DEGs were closely grouped in a number of pathways, including those involving ribosomal components, oxidative phophorylation, and cytochrome p450-mediated xenobiotic metabolism.It is worth noting that Supinoxin treatment as well as DDX5 knockdown resulted in significant alterations in oxidative phosphorylation (OXPHOS) as shown in Figure 7D.The OXPHOS pathway appears to be deactivated by Pathview with a p value of 0.0021.This deactivation is accompanied by the downregulation of 74 genes that are involved in the formation of complex I-V (Fig. 7F).It is worth noting that oxidative phosphorylation was also impacted upon DDX5 knock down in H69AR cells during our previous investigation 5 .Additionally, we conducted RT-qPCR on select genes (Fig. 7G) to examine the downregulation of DEGs in OXPHOS and mitochondrial function pathways following Supinoxin treatment.Upon Supinoxin treatment, we observed a significant downregulation of the relative steady state levels of DDX5, ACTB, SDHAF3, SUCLG1, COX5B, UQCRH, MDH1B, and NDUFA1 transcripts.Therefore, it can be concluded that Supinoxin treatment leads to the suppression of genes related to oxidative phosphorylation, which in turn causes mitochondrial dysfunction in H69AR SCLC cell lines.

Discussion
SCLC is a highly aggressive form of lung cancer, accounting for around 15% of all bronchogenic carcinomas, with an estimate of ~250,000 new cases diagnosed in the U.S. in 2022 (www.cancer.gov).SCLC is most strongly linked to smoking.It is worth noting that only a small percentage of cases, specifically 2%, are found in individuals who have never smoked 33,41,42 .The most common genetic alterations observed in SCLC involve the inactivation of TP53 and RB1, tumor-suppressor genes, in addition to copy-number gains of genes encoding MYC family members, receptor tyrosine kinases and their downstream effectors, chromatin remodeling enzymes, as well as Notch family proteins 33,43,44 .Individuals with lung-confined, localized SCLC cancer may opt for surgical resection to remove their primary tumor, which has been linked to enhanced survival rates 34,[45][46][47] .Unfortunately, close to 70% of SCLC patients are diagnosed with metastatic disease, frequently with macro-metastases in the brain, liver, lymph nodes, and bones 34 .The median survival rate for patients with SCLC is typically between 7-12 months following diagnosis [34][35][36]48 . Preious studies have shown that SCLC cells tend to spread through the lymphatic system and blood vessels at earlier stages compared to other types of lung cancer cells, making surgical resection less effective 49,50 .As a result, the treatment approach was redirected towards radiation and chemotherapy.It is worth noting that SCLC tumors exhibit a high sensitivity to chemotherapy initially [50][51][52] .
Nevertheless, the rapid recurrence of SCLC poses a significant challenge for oncologists in terms of treatment 53 .The etoposide/platinum (EP) combination was widely used as the standard treatment for SCLC until 2019.Additionally, it was shown that the addition of anti-programmed death-ligand 1 (anti-PD-L1) immunotherapy to EP chemotherapy resulted in enhanced survival 50,[54][55][56] .In the last three decades, there has been a lack of significant advancements in standard chemotherapy and radiation therapies for SCLC, with the exception of the recent approval of immune checkpoint inhibitors.These inhibitors have shown the ability to slightly prolong survival by only a few months 34,55,57,58 .Therefore, it is crucial to develop highly effective and innovative therapies for the treatment of SCLC.
The DEAD-box RNA helicases represent the most abundant category of enzymes within the RNA helicase family.These enzymes function as non-processive, ATP-dependent RNA-binding proteins, exerting their effects on remodeling secondary structures and/or RNA-protein complexes.They play crucial roles in various aspects of RNA biology, including transcription, translation, and RNA decay 59 .Interestingly, an increase in the expression of DEAD-box proteins has been associated with cellular transformation and is found in various types of cancer such as breast, colon, bone, and prostate cancer 29,60 .Earlier findings from our lab indicate that the RNA helicase DDX5 plays a crucial role in the invasive growth of SCLC.Knockdown of DDX5 leads to significant defects in mitochondrial respiration thereby altering the metabolism of SCLC cells so that they can no longer produce the necessary energy to sustain rapid growth 5 .The presence of impaired mitochondrial function suggests an increase in mitochondrial DNA (mtDNA) expression and the potential buildup of defective mitochondria in cancer cells 61 .DDX5 itself has been implicated in the development of various types of cancers 24,37,38,62,63 .
Reports suggest a strong connection between the development of various cancers and post translational modifications such as O-GlcNAcylation, sumoylation, and phosphorylation) of the DDX5 protein 13,64,65 .Previous investigations in our laboratory have demonstrated that, in contrast to the bronchial epithelial cell line HBEC-3KT (HBEC), the levels of DDX5 protein are significantly elevated in the chemosensitive H69 and chemoresistant H69AR SCLC cell lines 5 .Our current study has further confirmed our previous findings, showing a significant overexpression of the DDX5 protein in SCLC cell lines when compared to the human bronchial epithelial cell line HBEC-3KT.In addition, it was determined that increased protein stability of DDX5 in both SCLC cell lines contributes to the observed overexpression in the chemosensitive H69 and the chemoresistant H69AR SCLC cell lines.In addition, the results of IHC staining showed a significant increase in DDX5 expression in PDX SCLC tumors when compared to normal lung tissue.This finding emphasizes the importance of DDX5 in the development of SCLC.In addition, the observed over expression of DDX5 protein observed in both SCLC cell lines and PDX SCLC tumors are not associated with the elevated expression of DDX5 mRNA.In fact, transcriptome-wide studies have noted downregulation of DDX5 mRNA expression in lung cancer.During a study of 23 clinical small cell lung cancer (SCLC) samples from patients who had undergone pulmonary resection, researchers observed that the expression profile of the DDX5 gene was lower compared to that of a normal individual 39 .This data extends beyond small cell lung cancer, showing that DDX5 expression tends to be lower or remained similar in most tumor samples compared to normal tissues.This challenges the conventional belief that higher expression of transcripts always corresponds to increased protein levels.There is an apparent inverse correlation between the expression of DDX5 mRNA and its protein levels.The increased expression of DDX5 protein is associated with the greater stability of DDX5 protein in small cell lung cancer cells when compared to the normal bronchial epithelial cell line.We currently do not know why DDX5 is more stable in SCLC than normal, noncancerous cells or tissues.However, we speculate that post-translational modifications may be involved.Regardless, these studies show the importance of analyzing both the proteome as well as steady-state levels of mRNA to understand the mechanisms behind human disease states.
A small molecule inhibitor to DDX5, termed Supinoxin or RX-5902 11 , has been shown to abolish the growth and metastasis of triple negative breast cancer both in vitro and in vivo 12 .Studies indicate that treatment with Supinoxin led to apoptosis, G2/M cell cycle arrest, and aneuploidy in certain breast cancer cell lines 40 .We conducted a study to investigate the potential of repurposing Supinoxin as a treatment for SCLC.Our focus was on evaluating its efficacy in both cell line and patient derived xenograft mouse models.Our findings indicate that Supinoxin effectively hinders the growth of H69AR cell lines, as demonstrated by an IC50 value of 65.7 ±10.3 (Mean ± SD).Furthermore, it effectively suppresses the growth of H69AR cells in soft agar when combined with a concentration of 70 nM of Supinoxin.These findings indicate that Supinoxin has the potential to effectively suppress the growth of SCLC cell lines in vitro.Moreover, Supinoxin treatment also increases SCLC survivability and decreases tumor growth in a dose-dependent manner in in vivo mice models.However, Supinoxin does not completely eradicate the tumor, only slowing or pausing growth in both H69AR and PDX mouse models.
In addition, a series of experiments was conducted to explore if Supinoxin inhibits the proliferation of SCLC cells through a mechanism previously reported in breast cancer cells 40 .According to the proposed model 12,13 , Supinoxin disrupts the interaction between β-catenin and phosphor-DDX5, resulting in the release of β-catenin 13 .However, our experiments in H69AR cells neither detected a decrease in β-catenin levels nor a shift in the cellular localization of β-catenin.In addition, we found no alteration in the expression of β-catenin-dependent genes through RT-qPCR or at the protein level by Western blot.Next, we replicated the experiments in MDA-231, triple negative breast cancer cells.Again, there were no observed changes in β-catenin levels or re-localization from the cytoplasm, despite publications stating otherwise 13,40,65 .Therefore, we conclude that the current model for Supinoxin is incorrect.
In order to better understand the molecular mechanisms underlying the activity of Supinoxin, we performed RNA-seq on H69AR SCLC cell lines with and without Supinoxin treatment and analyzed the resulting differential gene expression patterns.
We compared it with our previously published RNA sequencing data of H69AR SCLC cells, both with and without DDX5 knockdown 5 .The findings were quite intriguing.The administration of Supinoxin results in the inhibition of genes associated with oxidative phosphorylation, leading to impaired mitochondrial function in H69AR SCLC cell lines (Figure 8).These findings align with our previous studies of DDX5 5 .Our research indicates that the genes affected by Supinoxin are a subset of those influenced by DDX5, as Supinoxin specifically targets a phosphorylated version of DDX5 12 .Even though we believe the current model for Supinoxin is wrong 12,13,40 , Supinoxin still impacts DDX5.We presently lack binding assay data to demonstrate how Supinoxin affects DDX5, aside from the prior model that indicates Supinoxin specifically binds to Y593 phosphorylated DDX5, indicating DDX5 as a Supinoxin cellular target 13 .However, our investigation revealed that Supinoxin treatment results in a decrease in DDX5 transcripts, indicating that Supinoxin treatment affects DDX5.We are now investigating the mechanism of Supinoxin.
The link between upregulation of ATP and cellular building block generation has been well established for decades as being a hallmark of cancers [66][67][68][69] .Several recent investigations have shown that oxidative phosphorylation (OXPHOS) is increased in different types of cancers, which could make them more susceptible towards OXPHOS inhibition.In addition, OXPHOS inhibition has been demonstrated to decrease the oxygen consumption rate which can help alleviate tumor hypoxia.This approach has also shown promise in certain cancers that have mtDNA mutations 70 .Cancer cells that lack mitochondrial DNA, resulting in a severe deficiency in oxidative phosphorylation, can only survive and thrive in vivo after implantation into mice if they are able to acquire intact mitochondria from the host [71][72][73] .In addition, research findings indicate that lung tumors possess a high level of oxidative activity, and that lung cancer development relies on OXPHOS [74][75][76][77] .According to a study, the regulation of mitochondrial respiration and OXPHOS by AIF (apoptosis-inducing factor) plays a role in the advancement of non-small cell lung cancer 77 .Multiple reports indicate that cancer stem cells (CSCs) heavily depend on oxidative phosphorylation (OXPHOS) rather than glycolysis [78][79][80][81] .In a study conducted by Lagadinou et al 80 , it was found that leukemia cells with CSCs exhibited a higher dependence on OXPHOS for their energy supply.It was proposed that inhibiting oxidative phosphorylation could effectively eliminate CSCs in the H446 small cell lung cancer cell line 81 .Our recent study reveals a decrease in Cytochrome c oxidase subunit 5B (COX5B) transcripts following Supinoxin treatment.It has been reported that COX5B could potentially play a significant role in predicting the prognosis of breast cancer 82 .Reducing the expression of COX5B in breast cancer cell lines has been found to inhibit cell growth and trigger cell senescence, resulting in an increase in the production of IL-8 and other cytokines 83 .Developing more effective therapeutic options requires a deeper understanding of the molecular mechanisms involved in the initiation, cellular transformation, progression, and establishment of SCLC chemoresistance.
Our studies found that Supinoxin (RX5902) has the ability to impede the proliferation of small cell lung cancer (SCLC).Moreover, Supinoxin effectively slows or temporarily halts tumor growth in both H69AR and PDX mouse models.We believe that the administration of Supinoxin in conjunction with immunotherapy or radiotherapy will have a significant impact on the management of SCLC.Our investigation holds significant value not only for the treatment of SCLC but also for targeted therapies in various types of cancers.Moreover, it suggests that exploration of RNA biology can lead to potential new targets for human disease intervention.

Cell Culture and transfection
H69AR cells were obtained from the American Type Culture Collection (ATCC) and cultured according to their instructions.The MDA-MB-231 cell line was a gift from Dr.
Michael Wendt (Purdue University) and was cultured in accordance with ATCC guidelines.Transfection was carried out using Lipofectamine 2000 reagent (Invitrogen, 11668027) at a confluency of 70-90%.

Growth analysis
Cells were seeded at a density of 10000 cells per well in 96-well plates (Corning, 3603).Supinoxin was dissolved in DMSO in order to prepare a stock solution of 5mM.
Following a 24-hour growth period, cells were treated with different concentrations of Supinoxin (0, 0.1, 1, 5, 10, 20, 40, 70, 100 and 1000 nM) diluted in appropriate growth media.The CyQUANT® direct cell proliferation kit (Thermo Fisher, C35011) was then used to measure the number of live cells.Relative fluorescence was calculated after subtracting background fluorescence from unlabeled cells.Soft agar assays were carried out as described 84 .GraphPad Prism 9 was used to determine the IC50 (half maximal inhibitory concentration) of Supinoxin.

RT-qPCR
TRIzol Reagent (Thermo 15596026) was used to extract total RNA from H69AR and MDA-MB-231 cells.cDNAs were then prepared from total RNA using the QuantiTect kit (Qiagen, 205310).To examine the expression levels of particular mRNAs, qPCR was carried out using SYBR green master mix (Applied Biosystems, 4309155) (Table 1 showing the list of primers used for the study).The relative expression level of specific RNAs was calculated using the Pfaffl method using the reference gene glyceraldehyde-3-phosphate dehydrogenase.

Microscopy
Cells were seeded onto glass coverslips coated with poly-L-lysine and given 24 hours to adhere.Following a 24-hour period (or as specified) of treatment with Supinoxin at increasing concentrations (0, 20, 70, or 100 nM), the cells were fixed with 1.6% formaldehyde in Phosphate Buffered Saline (PBS) (1:10 dilution of stock in PBS).Cells were then blocked with 2% BSA followed by incubation with primary antibodies for two hours.Primary antibodies include phosphor-DDX5 Y593(Abcam, ab62255), β-catenin (Thermo (Invitrogen) CAT-5H10).DNA was stained with 300 nM DAPI to serve as a nuclear marker.Cells were visualized using a Leica DM6 microscope with a 40X objective.

Histological staining
Histological staining was performed on paraffin-embedded samples of SCLC PDX and Normal lung tissues.Tissue sections were taken at a thickness of 4 micrometers using a microtome (Thermo HM355S).Sections were mounted on charged slides and dried on a 60 0 C oven.Slides were cleaned three times in xylene to deparaffinize the samples.Slides were then rehydrated by being submerged in decreasing concentrations of ethanol (100, 90, 70, 35%) followed by water Antigen unmasking was performed by pressure cooking slides for 20min in a buffer made from Tris Base (1M), EDTA (100mM), and Tween-20 (5%), with a pH adjusted to 9.0.Slides were then cleaned with TBST three times before being incubated in 3% hydrogen peroxide for 5 minutes.After an additional wash in TBST, sections were blocked using 2.5% goat serum in 1X TBST for 20min in a humidified chamber.Primary antibody diluted in blocking solution was added to each section followed by incubation for 30 minutes at room temperature in a humidified box.Sections were washed once more in TBST and then incubated with secondary antibody in blocking solution for 30 minutes at room temperature in a humidified chamber.ABC Reagent (Vectastain PK6100) and DAB Stock Solution (Vectastain, SK4100) kits were used in accordance with the manufacturer's instructions to stain the section.Hematoxylin (Sigma HHS15) was then used to counterstain the sections for 1 minute.The sections were then dehydrated by washes in increasing concentrations of ethanol (95 and 100% ethanol), followed by a xylene wash.Coverslips were then mounted onto slides using Permount (Fischer SP15-100).Images were taken using a Leica DM6 microscope with a 10X objective.

RNA-Seq and data analysis
Three biological replicates of H69AR cells treated with or without 70nM of Supinoxin were used for RNA sequencing.Total RNA extraction was performed using TRIzol Reagent (Thermo 15596026).Library construction and RNA sequencing were performed by Novogene Co., Ltd., Beijing, China.The libraries were sequenced using Illumina NovaSeq PE150 cassettes for paired-end sequencing with a 150-bp read length.Data quality control was conducted using the TrimGalore toolkit (version 0.4.4) (RRID:SCR_011847), with a minimum Phred score of 30 and a minimum read length of 50 bp.The quality trimmed reads were aligned to the human reference genome (GRCh38) obtained from the Ensembl database.The RNA-seq protocol included quality control (FastQC), trimming (Trimmomatic), alignment (HISAT2), and quantification (FeatureCounts) in a bash (Linux) environment.Differential expression analysis was performed using both DESeq2 and edgeR methods 85,86 .The analysis identified genes that were significantly differentially expressed (DEGs) based on a false discovery rate (FDR) threshold of < 0.05.The DESeq2 object included a pool of 2476 DEGs, which were used to investigate Gene Ontology (GO) and KEGG pathways.The oxidative phosphorylation pathway was visually represented using Pathview and the gage package, while GSEA conducted a comprehensive enrichment analysis of oxidative phosphorylation.The heatmap was created using Pheatmap and the ComplexHeatmap package in R-Studio.The pathways' direction was determined by utilizing the assigned Z-scores.The volcano plot was prepared using the Enhanced Volcano package in RStudio.In addition, the comparison table for the enrichment pathway was created using Tableau Desktop Public Version 2023.3.0,incorporating KEGG data from Supinoxin treatment and DDX5 knockdown 5 .

Tumor generation
Severely immunocompromised NRG mice (The Jackson Laboratory stock #007799) 10-16 weeks old (male or female) were used for all tumor implantations.Tumor volume was measured with digital calipers by taking 3 perpendicular measurements of LxWxH.Mice were monitored 2x/week for tumor growth and weight gain/loss and were euthanized when tumor volume reached 2000 mm 3 or if other humane criteria were met.H69AR cells still in log-phase growth were harvested from culture flasks and injected subcutaneously in the flank at 5x10 6 cells/mouse using a 25 ga needle.Cells were mixed 1:1 with Matrigel (50 µL:50 µL) for injection in groups of 5-6 mice.PDX tumors were implanted subcutaneously in the flank after harvesting tumors from donor mice.Donor tumors were minced into a paste with a razor blade, large fibrous chunks were removed, and remaining paste was mixed 1:1 with Matrigel (50 µL:50 µL) for injection with 14 ga needles in groups of 5-6 mice.Varying concentrations for Supinoxin were tested on H69AR cells on a 96-well plate for 24 hrs, after which cell viability was measured using a CyQUANT Direct Cell Proliferation Assay Kit (Invitrogen C35011).IC50 was calculated from four biological replicates.B. Soft agar colony formation assay was also performed with 0 and 70 nM to test the effect of Supinoxin on H69AR cells.H69AR cells were seeded on 0.3% agar layer in 6-well plates with 0 and 70 nM of Supinoxin.The cells were grown for 21 days with growth media added twice a week.
(β-Catenin-dependent gene) and DDX5 transcripts upon Supinoxin treatment in H69AR and MDA-MB-231 cells.Here, we also found out that the relative steady state levels of c-Myc, Cyclin-D1 and DDX5 transcripts remained significantly unaltered upon Supinoxin treatment.(Figure 4A,5A).

Figure 1 :
Figure 1: DDX5 protein is more stable in SCLC. A. Representative western blot images showing the expression of DDX5, RFC1 and GAPDH on HBEC-3KT, H69 and H69AR cells.B. The bar graphs depict the quantification of DDX5, RFC1 and GAPDH expression from three different biological replicates.These data represent the means and Standard deviation (SD) of three biological replicates.The following symbols represent the statistical significance of difference as reflected in the ranges of P values: *<0.05, **<0.005, and ***<0.001;ns, not significant.C. DDX5 gene expression profile of 23 clinical small cell lung cancer (SCLC) samples 39 from patients undergoing pulmonary resection in comparison to normal lung tissue obtained from Gene Expression Omnibus (GEO): GDS4794/225886_at.D. A bar plot illustrates the profile of DDX5 gene expression across all tumor samples and paired normal tissues.The data is obtained from Gene Expression Profiling Interactive Analysis (GEPIA): ENSG00000108654.11.E. RT-qPCR was used to quantify levels of DDX5 transcripts on HBEC-3KT, H69 and H69AR samples, which were normalized to the expression levels of glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA.These data represent the mean and standard deviation (SD) of three biological replicates.The following symbols represent

Figure 2 :
Figure 2: Inhibition of H69AR cell growth by Supinoxin.A.Varying concentrations for Supinoxin were tested on H69AR cells on a 96-well plate for 24 hrs, after which cell viability was measured using a CyQUANT Direct Cell Proliferation Assay Kit (Invitrogen C35011).IC50 was calculated from four biological replicates.B. Soft agar colony formation assay was also performed with 0 and 70 nM to test the effect of Supinoxin on H69AR cells.H69AR cells were seeded on 0.3% agar layer in 6-well plates with 0 and 70 nM of Supinoxin.The cells were grown for 21 days with growth media added twice a week.

Figure 3 :Figure 4 :
Figure 3: Supinoxin inhibits tumor growth and increases the survival of mice with H69AR-based tumors and patient-derived xenograft (PDX) SCLC tumors.A. Immunocompromised NOD-Rag1null IL2rgnull (NRG) mice bearing with H69AR xenograft tumors were treated with 17.5, 35, or 70mg/kg Supinoxin or vehicle only for 25 days to identify the best dose for tumor growth inhibition in the absence of significant toxicity based on weight gain.B. Overexpression of DDX5 in Patient-derived Xenograft (PDX) Small cell Lung Cancer Samples (SCLCs) used to generate PDX tumors.Histological staining was performed on SCLC PDX tumors and normal lung tissues.Images were taken using a Leica DM6 microscope with 10X objective.Brown staining indicates DDX5 staining.C. Immunocompromised NRG mice bearing patient-derived xenograft (PDX) tumors were administered Supinoxin at 70mg/kg as a slurry using saline with 10% DMSO as the vehicle.Supinoxin or vehicle was administered for a duration of 8 weeks, to assess the survival curve in each group.D. Mice with PDX

Figure 5 :
Figure 5: The expression of c-Myc, DDX5 and β-Catenin does not change significantly upon treatment with various concentrations of Supinoxin on MDA-MB-231 cells.A. RT-qPCR on MDA-MB-231 samples treated with various concentrations of Supinoxin.RT-qPCR was used to quantify levels of DDX5, Cyclin D1 and c-Myc transcripts, which were normalized to the expression levels of GAPDH mRNA.These data represent the means and Standard deviation (SD) of three biological replicates.The following symbols represent the statistical significance of difference as reflected in the ranges of P values: *<0.05, **<0.005, and ***<0.001;ns, not significant.B. Representative western blot images showing the expression of c-Myc, DDX5, β-Catenin and β-Actin following Supinoxin treatment on MDA-MB-231 cells.C,D,E.The bar graphs depict the quantification of c-Myc, DDX5 and β-Catenin expression from three-four different biological replicates.These data represent the means and Standard deviation (SD) of three biological replicates.The following symbols represent the statistical significance of difference as reflected in the ranges of P values: *<0.05, **<0.005, and ***<0.001;ns, not significant.

Figure 6 :
Figure 6: Supinoxin has no effect on the localization of β-Catenin or pDDX5 in H69AR and MDA-MB-231 cells.A. The localization of β-Catenin did not change in the presence or absence of Supinoxin on H69AR cells or MDA-MB-231 cells.The cells were seeded onto coverslips and allowed to grow overnight.Supinoxin was then added and incubated on cells for 24hrs, after which cells were fixed.The cells were then prepared for immunofluorescence microscopy to visualize the localization of β-Catenin.Cells were visualized using Leica DM6 microscope using 40X objective.B. The localization of pDDX5 does not change in the presence of various concentrations of Supinoxin on H69AR cells and MDA-MB-231 cells.pDDX5 is shown in green and DAPI stain is shown in blue.H69AR cells and MDA-MB-231 cells were seeded onto

Figure 7 :
Figure 7: RNA-Seq analysis reveals downregulated mitochondrial function following Supinoxin treatment.A. Venn diagram illustrating the total number of transcripts upregulated and downregulated with Supinoxin treatment and DDX5 knockdown.Venn diagrams were generated using Venn Diagram package in RStudio-2023.12.0 B. Heat map showing the differential expression patterns of various transcripts that are affected by Supinoxin treatment.Red denotes upregulation, whereas blue denotes downregulation.The log2 fold change values are used to indicate the direction of gene expression.Assigned Z-scores were used to enhance clusters of genes with expression patterns across the samples and to understand how genes work

Figure 8 :
Figure 8: A model figure depicting the mechanism of Supinoxin in Small-cell lung cancer.Supinoxin interacts with DDX5 and down-regulates genes involved in oxidative